Project Summary The major transcription factor that drives responses to type I interferon (IFN-I) is ISGF3, comprised of tyrosine- phosphorylated STATs 1 and 2 and the DNA binding protein IRF9. The threonine phosphorylation of STAT2 on T387 and T404 profoundly affects the ability of IFN-I to drive gene expression and dependent biological functions. Furthermore, STAT2 lacking tyrosine phosphorylation (U-STAT2) enhances the expression of NF-?B target genes that encode several chemokines and pro-inflammatory cytokines (for example, IL-6), and also inhibits the ability of IFN-? to activate the tyrosine phosphorylation of STAT1 and downstream gene expression. These non-canonical functions of U-STAT2 are also regulated by its phosphorylation on T387 and T404. Dramatic effects are seen with mice carrying homozygous T-A mutations at each of these two sites. The defective response to IFN-I in T403A mice causes them to be highly susceptible to infections with vesicular stomatitis virus (VSV) or Herpes Simplex virus (HSV). (The numbers for mice are T386 and T403.) The responses of T403A mice to inflammatory stimuli are also greatly compromised. Ulcerative colitis is a chronic disease in which the lining of the colon becomes inflamed and develops ulcers. In the dextran sulfate sodium (DSS)-induced model of ulcerative colitis, T403A/T403A knockin mice, in which this site cannot be phosphorylated, are very much more sensitive than WT/WT mice to DSS-induced mortality and weight loss. Sepsis and septic shock are the leading causes of death in non-coronary intensive care units. We have shown that the immune response in sepsis transitions from an early/hyper-inflammatory to a late/hypo-inflammatory, immunosuppressive phenotype that is associated with multiple organ dysfunction involving lung, heart, kidney and liver. Furthermore, prolonged hypo-inflammation is associated with impaired pathogen clearance. Compared with wild- type controls, T403A/T403A mice are protected from, and T386A/T386A mice are sensitized to challenge with lipopolysaccharide (LPS), an inducer of septic shock, and the induction of IFN-I and IL-6 expression is suppressed in T403A/T403A mice. Our primary objectives follow. 1) We want to understand in detail how the phosphorylation of STAT2 on these two threonine residues is regulated (which activating signals? which kinases? which phosphatases?). 2) We want to understand in detail how pro-inflammatory mechanisms are affected by STAT2 threonine phosphorylation. 3) We want to use this information to develop novel therapeutic approaches to inflammatory responses to commensal bacteria, especially colonic inflammation and septic shock. We anticipate that we can parlay improved understanding of the roles of threonine phosphorylation of STAT2 in inflammation and infection into novel therapeutic approaches, and that the discovery of novel approaches to ameliorate bacterially-induced acute inflammation may also be helpful in other inflammatory conditions.